ABSTRACT Cellular membranes are critical components of all free-living organisms. However, knowledge of their biosynthesis and modification has been hindered by the hydrophobicity engendered by their lipid constituents. Lipids are synthesized and modified primarily by integral membrane enzymes embedded, at least in part, in the bilayer. However, the atomic-level details of lipid/enzyme interactions and the determinants of their specificity remain poorly understood. Here we present preliminary structural and functional characterization for three distinct families of integral membrane lipid-modifying enzymes. Two of the representative crystal structures have a bound lipid ligand, tripling the number of examples of membrane-embedded enzymes visualized with their cognate hydrophobic substrate. Each of the structures is a first representative of a large enzyme family: (1) GtrB, a polyisoprenyl phosphate glycosyltransferase attaches glucose to a lipid carrier for membrane translocation and a glycosyl donor for subsequent reactions. This reaction represents the first step in all protein glycosylation and glycosylation of the cell wall. (2) ArnT uses sugar-charged donors produced by GtrB-like enzymes, and transfers the saccharide to lipid A on the cell surface of bacteria, altering antibiotic resistance properties. (3) We present the structure of the Renibacterium salmoninarum phosphatidylinositol-phosphate (PIP) synthase ? an enzyme required for inositol-lipid synthesis ? with a bound CDP-diacylglycerol substrate. This enzyme is a member of the CDP-alcohol phosphotransferase family (CDP-APs), which catalyze the defining step in glycerophospholipid biosynthesis across all kingdoms of life. We will explore substrate recognition by these enzymes with a combination of experimental approaches including x-ray crystallography and structure-guided mutagenesis coupled to functional readouts in bacteria, yeast, and zebrafish. The overall goal of this proposal is to understand the basic principles of substrate recognition in lipid biosynthesis and modification reactions.